plants Article A Survey of Trunk Disease Pathogens within Citrus Trees in Iran Nahid Esparham 1, Hamid Mohammadi 1,* and David Gramaje 2,* 1 Department of Plant Protection, Faculty of Agriculture, Shahid Bahonar University of Kerman, Kerman 7616914111, Iran; [email protected] 2 Instituto de Ciencias de la Vid y del Vino (ICVV), Consejo Superior de Investigaciones Científicas, Universidad de la Rioja, Gobierno de La Rioja, 26007 Logroño, Spain * Correspondence: [email protected] (H.M.); [email protected] (D.G.); Tel.: +98-34-3132-2682 (H.M.); +34-94-1899-4980 (D.G.) Received: 4 May 2020; Accepted: 12 June 2020; Published: 16 June 2020 Abstract: Citrus trees with cankers and dieback symptoms were observed in Bushehr (Bushehr province, Iran). Isolations were made from diseased cankers and branches. Recovered fungal isolates were identified using cultural and morphological characteristics, as well as comparisons of DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region, translation elongation factor 1α, β-tubulin, and actin gene regions. Dothiorella viticola, Lasiodiplodia theobromae, Neoscytalidium hyalinum, Phaeoacremonium (P.) parasiticum, P. italicum, P. iranianum, P. rubrigenum, P. minimum, P. croatiense, P. fraxinopensylvanicum, Phaeoacremonium sp., Cadophora luteo-olivacea, Biscogniauxia (B.) mediterranea, Colletotrichum gloeosporioides, C. boninense, Peyronellaea (Pa.) pinodella, Stilbocrea (S.) walteri, and several isolates of Phoma, Pestalotiopsis, and Fusarium species were obtained from diseased trees. The pathogenicity tests were conducted by artificial inoculation of excised shoots of healthy acid lime trees (Citrus aurantifolia) under controlled conditions. Lasiodiplodia theobromae was the most virulent and caused the longest lesions within 40 days of inoculation. According to literature reviews, this is the first report of L. theobromae and N. hyalinum on citrus in Iran. Additionally, we report several Phaeoacremonium species, S. walteri, Pa. pinodella and C. luteo-olivacea on citrus trees for the first time in the world. Keywords: bscogniauxia; botryosphaeriaceae; cadophora; citrus dieback; colletotrichum; phaeoacremonium 1. Introduction Iran is the sixth largest Citrus producer, accounting for 3.3% of the world’s Citrus production, which yielded 4.1 million tons in 2016 [1]. A total of 276,000 ha of various Citrus species are cultivated in Iran, including sweet orange (Citrus sinensis L.), acid lime (C. aurantifolia (Christm.) Swingle), sour orange (C. aurantium L.), mandarin (C. reticulata Blanco), lemon (C. limon (L.) Osbeck), and grapefruit (C. paradisi Macfad). The most important producing regions in Iran are Mazandaran, Fars, Hormozgan, Giroft, and Kahnouj. Fungal trunk diseases have been studied in detail in grapevine, which are the main biotic factor limiting vineyard productivity and longevity [2]. However, recent findings of high incidence in stone and pome fruits, small fruits, nut crops, citrus, and olive worldwide highlight the need for a focus on this novel group of hosts [3]. Trunk diseases are caused by a broad range of taxonomically unrelated fungi that primarily infect wood hosts through winter pruning wounds, thus colonizing the vascular tissues. Members of the families Botryosphaeriaceae, Togninaceae, Diatrypaceae, Diaporthaceae, as well as several basidiomycetes are included in this group of fungi. Members of Diatrypaceae Plants 2020, 9, 754; doi:10.3390/plants9060754 www.mdpi.com/journal/plants Plants 2020, 9, 754 2 of 20 (Xylariales) can often be observed on dead wood and bark of a wide range of plant species around the world. Nevertheless, some species of this family are reported as putative plant pathogens on fruit, ornamental, and forest trees [4–8]. Some species of Eutypella have been previously isolated from citrus species, including Citrus limon, C. paradisi, C. maxima, and C. aurantium in Australia, Argentina, Brazil, Coted’Ivoire, Philippines, and USA [9–14]. Diatrypaceae spp. were also isolated from citrus trees in Australia [15]. Species of Botryosphaeriaceae have a cosmopolitan distribution and have been associated with numerous plant species worldwide [16–18]. Many species of the genera Lasiodiplodia [19–23], Diplodia, Dothiorella, Neofusicoccum [20], and Neoscytalidium [14,20] have been previously reported to affect citrus trees. Togniniaceae (Togniniales), with the well-known asexual morph genus Phaeoacremonium, is another family of fungi traditionally associated with dieback, canker, and yellowing of various fruit, forest, and ornamental trees, worldwide [24–31]. To date, 56 Phaeoacremonium species have been identified from woody hosts [32]. The most prevalent Phaeoacremonium species isolated from woody hosts are P. minimum, followed by P. parasiticum [33]. Dieback and related disease symptoms have been achieved by inoculating Phaeoacremonium species onto several hosts such as Prunus spp., kiwifruit, oak, and grapevine [33,34]. Phaeoacremonium species isolated from grapevine have been intensively studied because of their involvement in two trunk diseases, Petri disease in young vines and esca in mature vines [32,35]. To our knowledge, there are no reports of Phaeoacremonium species affecting Citrus spp. In spring 2014, a severe decline of citrus trees was noticed in some orchards in Bushehr (Bushehr province, Iran). External disease symptoms included chlorosis of leaves, defoliation, branch and shoot cankers, and dieback. Internal wood symptoms ranged from brown to black wood streaking and black spots to wedge-shaped necrosis, irregular wood discoloration, central necrosis, and arch-shaped necrosis. Many fungi associated with trunk diseases have been isolated from several woody hosts in Iran, including grapevine [36,37], pome and stone fruit trees [28,38], and ornamental and forest trees [29,31,39,40]. However, little information is presently available on the causal agents of the severe decline of citrus trees in Iran. Therefore, the aim of this study was to investigate the etiology of fungal trunk diseases associated with wood necrosis of citrus trees in Iran and to determine their pathogenicity. 2. Results 2.1. Field Survey and Diversity of Disease Symptoms In this study, wood samples were collected from lime (46 trees), sweet lemon (23 trees), sweet orange (22 trees), mandarin (eight trees), sour orange (four trees) and lemon (three trees). Citrus trees showed various external disease symptoms, including yellowing, canker, defoliation, dieback, cracking of the bark associated with gumming, and sooty cankers (form a black powder underneath the bark). Examination of infected branches from symptomatic trees revealed different types of wood discoloration in cross-sections, black to brown streaking in the wood, wedge-shaped necrosis, black spots, irregular wood discoloration, central necrosis, and arch-shaped necrosis (Figure1). Plants 2020, 9, x FOR PEER REVIEW pycnidia, on pine needles within 15–35 days. Conidia were pigmented or hyaline. These isolates belonged to the genera Lasiodiplodia, Neoscytalidium, and Dothiorella. Twelve isolates of the phialides fungus were identified as Cadophora sp. These isolates formed flat, felty, and black-olivaceous and white to gray colonies on PDA, and their conidia were ellipsoid or elongate. Cultural and morphological characteristics observed were similar with the description of the Cadophora spp. [43,44]. Based on morphological characteristics, the remaining isolates were classified to Colletotrichum, Peyronellea, Pestalotiopsis, Fusarium, Microsphaeropsis, Alternaria, Trichoderma, Plants 2020, 9, 754 3 of 20 Paecilomyces, Aspergillus, Penicillium, Phoma, Biscogniauxia, and Stilbocrea genera. Figure 1. Diversity of external (A–D) and internal (E–I) trunk disease symptoms on Citrus species in Figure 1. Diversity of external (A–D) and internal (E–I) trunk disease symptoms on Citrus species in Iran (A) a severe dieback on Citrus sinensis; (B) two cankers on a trunk of a C. sinensis tree indicated Iran (A) a severe dieback on Citrus sinensis;(B) two cankers on a trunk of a C. sinensis tree indicated by by arrows; (C) an extended canker on the branch of Citrus aurantifolia indicated by arrow; (D) arrows; (C) an extended canker on the branch of Citrus aurantifolia indicated by arrow; (D) gummosis on Citrus limetta;(E) cross-section of a healthy branch of C. aurantifolia;(F) central necrosis on C. sinensis; (G) Co-occurrence of brown wood streaking (black arrow), wedge-shaped necrosis (white arrow) and irregular wood necrosis (red arrow) on C. sinensis;(H) Arch-shaped necrosis on C. aurantifolia; (I) a young wedge shaped necrosis on Citrus reticulata;(J) Co-occurrence of wedge-shaped necrosis is indicated by the white arrow and black spots are indicated by the black arrow on the C. sinensis. Plants 2020, 9, 754 4 of 20 2.2. Fungal Isolation and Morphological Identification In this survey, 326 fungal isolates were collected from citrus trees (Table1). According to colony appearance, culture characteristics, and microscopic structures, the main fungal isolates were classified as Phaeoacremonium spp., Botryosphaeriaceae spp. Cadophora sp., Colletotrichum spp., Peyronellaea sp., Phoma spp., and Biscogniauxia mediterranea. Thirty-nine isolates (11.96% of total isolates) were identified as Phaeoacremonium species and characterized by beige to medium brown flat slow-growing cultures on potato dextrose agar (PDA; Merck, Darmstadt, Germany) and on malt extract agar (MEA; 2% malt extract, Merck, Darmstadt, Germany). Septate